CN103328643A - Method for producing cadaverine - Google Patents
Method for producing cadaverine Download PDFInfo
- Publication number
- CN103328643A CN103328643A CN2011800593548A CN201180059354A CN103328643A CN 103328643 A CN103328643 A CN 103328643A CN 2011800593548 A CN2011800593548 A CN 2011800593548A CN 201180059354 A CN201180059354 A CN 201180059354A CN 103328643 A CN103328643 A CN 103328643A
- Authority
- CN
- China
- Prior art keywords
- cadaverine
- gene
- flora
- shaped
- bar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 claims abstract description 15
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01018—Lysine decarboxylase (4.1.1.18)
Abstract
Disclosed is a novel method for producing cadaverine, said method being able to produce cadaverine at a higher yield and a greater efficiency than production methods by means of conventional fermentation methods. The method for producing cadaverine includes the culturing of coryneform bacteria that have the ability to produce cadaverine and that have resistance to 2,2'-thiobis(ethylamine). Preferably, the coryneform bacterial have lysine decarboxylase activity, and preferably, the coryneform bacteria have homoserine auxotrophy and/or S-(2-aminoethyl)-L-cysteine resistance.
Description
Technical field
The present invention relates to make with the bar-shaped flora with cadaverine throughput the method for cadaverine.
Background technology
Cadaverine has two amine structures, and another name is called 1,5-pentamethylene diamine or pentamethylene diamine etc.Recently, cadaverine receives publicity as the raw material monomer of polymeric amide, and therefore expectation is a large amount of produces.Manufacture method as cadaverine, the known fermentation method that utilizes bar-shaped flora, particularly, known have: make the method for cadaverine by the fermentation of following bar-shaped flora, described bar-shaped flora has cadaverine throughput and as the synthesis capability of the Methionin of cadaverine precursor substance strengthened (refer to Patent Document 1～4, non-patent literature 1); Perhaps make the method for cadaverine by the fermentation of following bar-shaped flora, described bar-shaped flora is owing to the gene copy number increase of lysine decarboxylase gene makes lysine decarboxylase activity strengthened (referring to Patent Document 5).
The prior art document
Patent documentation
Patent documentation 1: TOHKEMY 2004-222569 communique
Patent documentation 2: TOHKEMY 2002-223770 communique
Patent documentation 3:WO2007/113127 number
Patent documentation 4:WO2008/101850 number
Patent documentation 5:WO2008/092720 number
Non-patent literature
Non-patent literature 1:Stefaine Kind, Metabolic engineering. (metabolic engineering) 12,341-351, (2010)
Summary of the invention
The problem that invention will solve
Problem of the present invention is to develop Billy makes cadaverine with traditional fermentation method the cadaverine manufacturing process that method is more effective and productive rate is higher.
The means of dealing with problems
The inventor finds, has the ability of producing cadaverine and the bar-shaped flora that 2,2'-thiobis (ethamine) has a resistance be can be used as cadaverine to produce bacterium, thereby cause of the present invention finishing.
That is, the invention provides following (1)～(5).
(1) a kind of manufacture method of cadaverine comprises cultivating to have the ability of producing cadaverine and the bar-shaped flora that 2,2'-thiobis (ethamine) is had resistance.
(2) according to the manufacture method of (1) described cadaverine, described bar-shaped flora is to 2 more than the 250mM, and 2'-thiobis (ethamine) has resistance.
(3) according to the manufacture method of (1) or (2) described cadaverine, described bar-shaped flora has the lysine decarboxylase activity.
(4) manufacture method of each described cadaverine in the basis (1) to (3), the group that the free corynebacterium of described corynebacterium mass selection and brevibacterium sp consist of.
(5) according to the manufacture method of each described cadaverine in (1) to (4), described bar-shaped flora has homoserine auxotrophy and/or S-(2-aminoethyl)-Cys resistance.
The effect of invention
According to the present invention, compare with the method for utilizing traditional fermentation method to make cadaverine, can be more effective and more high productivity make cadaverine.
Embodiment
As mentioned above, in the method for the invention, used bar-shaped flora.Bar-shaped flora is the aerobic Gram-positive bacillus, is classified as traditionally brevibacterium sp, but also be included in now unified in the bacterium of corynebacterium (Int.J.Syst., Bacteriol., (1981) 41, p.225).In addition, comprise the brevibacterium sp bacterium that approaches very much with corynebacterium.
As the example of so bar-shaped flora, can list: Corynebacterium acctoacidophlum (Corynebacterium acetoacidophylum), corynebacterium acetoglutamicum (Corynebacterium acetoglutamicum), corynebacterium alkanolyticum (Corynebacterium alkanolyticum), corynebacterium callunae (Corynebacterium callunae), Corynebacterium glutamicum (Corynebacterium glutamicum), lily hedysarum scoparium bacillus (Corynebacterium lilium), corynebacterium melassecola (Corynebacterium mellassecola), hot corynebacterium ammoniagenes (Corynebacterium thermoaminogenes), effective excellent bacillus (Corynebacterium efficiens), man of great strength's rod bacillus (Corynebacterium herculis), fork tyrothricin (Brevivacterium divaricatum), brevibacterium flavum (Brevivacterium flavum), Block レ PVC バ Network テ リ ウ system イ Application マ リ オ Off ィ ラ system (Brevivacterium immariophilum), brevibacterium lactofermentum (Brevivacterium lactofermentum), rose-colored tyrothricin (Brevivacterium roseum), Brevibacterium saccharolyticum (Brevivacterium saccharolyticum), give birth to sulphur tyrothricin (Brevivacterium thiogenitalis), produce ammonia rod bacillus (Corynebacterium ammoniagenes), white tyrothricin (Brevivacterium album), Block レ PVC バ Network テ リ ウ system セ リ ヌ system (Brevivacterium cerinum), have a liking for ammonia microbacterium (Microbacterium ammoniaphilum).
In addition, concrete bacterial strain as each bar-shaped flora, can enumerate: Corynebacterium acctoacidophlum ATCC13870, the bar-shaped flora ATCC15806 of vinegar L-glutamic acid, corynebacterium alkanolyticum ATCC21511, corynebacterium callunae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13020, ATCC13060, lily hedysarum scoparium bacillus ATCC15990, corynebacterium melassecola ATCC17965, effective excellent bacillus AJ12340(deposit number: FERMBP-1539), man of great strength's rod bacillus ATCC13868, fork tyrothricin ATCC14020, brevibacterium flavum ATCC13826, ATCC14067, AJ12418(deposit number: FERMBP-2205), Block レ PVC バ Network テ リ ウ system イ Application マ リ オ Off ィ ラ system (Brevivacterium immariophilum) ATCC14068, brevibacterium lactofermentum ATCC13869, rose-colored tyrothricin ATCC13825, Brevibacterium saccharolyticum ATCC14066, give birth to sulphur tyrothricin ATCC19240, produce ammonia rod bacillus ATCC6871, ATCC6872, white tyrothricin ATCC15111, Block レ PVC バ Network テ リ ウ system セ リ ヌ system (Brevivacteriumcerinum) ATCC15112 has a liking for ammonia microbacterium ATCC15354.
Above-mentioned bar-shaped flora can be bought from (for example) American type culture collection (ATCC) and obtain.In ATCC, each bacterial strain is accompanied with corresponding registration number, and this registration number is documented in the catalogue of ATCC, can buy with reference to this registration number and obtain each bacterial strain.
In the present invention, as the bar-shaped flora with the ability of producing cadaverine, the preferred bar-shaped flora that has the lysine decarboxylase activity by the polynucleotide that import the coding lysine decarboxylase from the outside that uses.As long as have the lysine decarboxylase activity, namely can take Methionin as raw material, by being carried out decarboxylation, it produce cadaverine.
Lysine decarboxylase is preferably the 1B decarboxylase.In addition, source about lysine decarboxylase also is not particularly limited, but preferred the use derives from (for example) salt tolerant genus bacillus (Bacillus halodurans), subtilis (bacillus subtilis), colon bacillus (Escherichia coli; Intestinal bacteria), ruminate Selenomonas (Selenomonas ruminamtium), vibrio cholerae (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahaemolyticus), streptomyces coelicolor (Streptomyces coelicolor), hair streptomycete (Streptomyces pilosus), corrode Aitken bacterium (Eikenella corrodens), bite amino acid Eubacterium (Eubacterium acidaminophilum), Salmonella typhimurtum (Salmonella typhimurium), hafnia alvei (Hafnia alvei), Neisseria meningitidis (Neisseria meningitidis), thermoplasma acidophilum (Thermoplasma acidophilum), perhaps the lysine decarboxylase of P.abyssi (Pyrococcus abyssi) more preferably uses and derives from the colibacillary lysine decarboxylase that security is confirmed.The aminoacid sequence of these lysine decarboxylases (sometimes being called as " LDC ") and the base sequence that it is encoded is registered in the database (GenBank).The base sequence that derives from colibacillary LDC gene that for example, can preferably use in the present invention is registered as GenBank Accession No.M76411.
The gene of the coding lysine decarboxylase that uses among the present invention can design nucleotide sequence according to the codon usage frequency of employed bar-shaped flora again.Need to prove, as mentioned above, derive from above-mentioned each biological lysine decarboxylase gene and be registered in the database (GenBank), retrieve the base sequence that namely can easily learn each LDC gene as keyword with biological name and lysine decarboxylase.
Gene as the coding lysine decarboxylase, in having the scope of its function, except the natural LDC gene of above-mentioned each biological species, also be included in each base sequence of these LDC genes, the displacement, disappearance, insertion of one or more bases occurs or add after polynucleotide.Here, " a plurality of " are generally 1 to 40 degree, and preferred 1 to 30, more preferably 1 to 20, particularly preferably 1 to 10, most preferably 1 to 5.In addition, as the gene of above-mentioned coding lysine decarboxylase, in having the scope of its function, can list the polynucleotide with all or a part of hybridize under stringent condition of the polynucleotide that consist of this gene or its complementary strand.Here, " polynucleotide of hybridize under stringent condition " refer to that (for example) will be selected from the original base sequence at least 20 arbitrarily, is preferably 25, more preferably one or more nucleotide sequences at least 30 continuous sequences are as probe, use known hybridization technique (Current Protocols I Molecular Biology edit.Ausbel et at., (1987) Publish.John Wily﹠amp; Sons Section6.3-6.4) etc. the nucleotide sequence of hybridizing.Herein as stringent condition, for example in the presence of 50% methane amide, hybridization temperature be 37 ℃, as stricter condition be 42 ℃, when further stricter condition is 65 ℃, SSC solution (composition of the SSC solution of 1 times of concentration: 150mM sodium-chlor, 15mM Trisodium Citrate) washing by with 0.1 to 2 times of concentration can realize hybridization thus.In addition, as the polynucleotide of above-mentioned coding lysine decarboxylase, in having the scope of its function, also can for sequence identity usually more than 85%, be preferably more than 90%, more preferably more than 95%, polynucleotide more than 99% more preferably.Herein, " sequence identity " refers to carry out that sequence 2 base sequences are relatively arranged so that the base of two sequences as much as possible consistent (inserting as required the gap), with consistent base number divided by whole base numbers and the numerical value that represents with percentage.When the base number of 2 sequences not simultaneously, divided by the long side's of sequence base number.The conforming software of the sequence of calculation is well-known, and is freely open on the internet.The gene of such coding lysine decarboxylase also can be obtained beyond original host, can also obtain by the gene that obtains from original host being carried out the well-known external sudden change processing of those skilled in the art or site specific sudden change processing.
To bar-shaped flora import the coding lysine decarboxylase gene (below sometimes be also referred to as " lysine decarboxylase gene " or " LDC gene ", yet as mentioned above, it is not limited to natural gene) time, the lysine decarboxylase gene can be maintained in being maintained at extrachromosomal plasmid of bar-shaped flora etc., also can be incorporated in the karyomit(e) of bar-shaped flora and is maintained.
When integrating the lysine decarboxylase gene in the karyomit(e) of bar-shaped flora, can utilize transposon, the lysine decarboxylase gene be imported in the karyomit(e) of bar-shaped flora by homologous recombination or the transfer ability of himself.Need to prove, the structure of the gene order that imports or its confirmation method can be finished by Protocols in Molecular Biology well-known to those skilled in the art, such as can be with reference to the Molecular Cloning:A Laboratory Manual of Sambrook etc., Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning:A Practical Approach, Volumes I and II (D.N.Glovered.1985); F.M.Ausubel etc. (eds), Current Protocols in Molecular Biology (1994) John Willey﹠amp; Sons, Inc.; PCR Technology:Principles and Application for DNA Amplication, H.Erlich, ed., Stockton Press, etc.
The said gene construct is not particularly limited to the introduction method in the bar-shaped flora, for example, can passes through protoplasm body (Gene, (1985), 39, p.281-286), electroporation (Bio/Technology, (1989), 7, the importing such as 1067-1070).
Need to prove, importing external LDC gene and having bar-shaped flora that cadaverine produces ability is known (with reference to following reference example) as (for example) patent documentation 1 is described.
Next, specify the bar-shaped flora that 2,2'-thiobis (ethamine) is had resistance.
2,2'-thiobis (ethamine) is by structural formula NH
2-(CH
2)
2-S-(CH
2)
2-NH
2The strong basicity chemical substance of expression.In the present invention, 2,2'-thiobis (ethamine) being had resistance refers in the substratum that contains 2,2'-thiobis (ethamine) than the wild type strain faster character of growing.Particularly, containing 2 more than the 200mM, on the plate culture medium of 2'-thiobis (ethamine), wild type strain can't form bacterium colony, but for the bacterial strain that formed bacterium colony at the 2nd～3 day, can judge that it has 2,2'-thiobis (ethamine) resistance.Need to prove, have no particular limits in the concentration that plate culture medium demonstrates the thiobis (ethamine) of resistance for bar-shaped flora, but more than the preferred 250mM, more preferably more than the 300mM, more than the further preferred 400mM.
To being used for estimating 2 of bar-shaped flora, 2'-thiobis (ethamine) resistance contain 2, the substratum of 2'-thiobis (ethamine) has no particular limits, get final product so long as can cultivate the substratum of bar-shaped flora, but preferably do not add the minimum medium of amino acid etc., more preferably the minimum medium shown in the table 1.
[table 1]
Method as give from 2,2'-thiobis (ethamine) resistance to bar-shaped flora has no particular limits, for example, as long as make the karyomit(e) of maternal plant produce certain sudden change.The technology that imports sudden change in the karyomit(e) has: the irradiation by UV, laser etc. produces the method for sudden change; And the method etc. of using the mutagenic compound such as ethyl sulfonic acid methyl esters (EMS), nitrosoguanidine (NTG), 4-Dimethylaminobenzene diazosulfonic acid sodium (DAPA).In addition, also can utilize the spontaneous mutation that when cultivating bar-shaped flora, produces.Need to prove, has the ability of producing cadaverine and to 2 as providing, 2'-thiobis (ethamine) has the method for the bar-shaped flora of resistance, can give described resistance to the bar-shaped flora with the ability of producing cadaverine, in addition, also can give in the above described manner the ability of producing cadaverine to the bar-shaped flora of having given this resistance.Need to prove, said mutation is brought out to process to bring out under the known condition that adopts in the processing in the sudden change of microorganism and is got final product, and for example, when UV shone, irradiation dose depended on the distance with light source, yet usually carries out 10 seconds～30 minutes irradiation.In addition, when processing with various mutagenic compound such as above-mentioned NTG, concentration for the treatment of is (for example) 100 μ g/ml～2 μ g/ml, is preferably 300 μ g/ml～1.3 μ g/ml, can cultivate in the nutrient solution of this concentration by (for example) and process in 12 hours～48 hours.
After processing is brought out in this sudden change, above-mentioned for estimating 2, cultivate on the substratum of 2'-thiobis (ethamine) resistance, and by to having obtained 2, the bacterial strain of 2'-thiobis (ethamine) resistance screens, can obtain thus to have the bar-shaped flora of 2,2'-thiobis (ethamine) resistance.Need to prove, as specifically described in the following enforcement, can obtain a plurality ofly having obtained 2 by processing by NTG, the bacterial strain of 2'-thiobis (ethamine) resistance is learnt, bring out processing by sudden change, can produce to reproducibility 2,2'-thiobis (ethamine) resistant strain.
In addition, the bar-shaped flora that uses among the present invention is preferably the mutant strain that the Methionin synthesis capability is improved.The mutant strain that is improved as the Methionin synthesis capability for example, can utilize the mutant strain of having removed the feedback inhibition that causes because of 1B or L-threonine.Preparation method as these mutant strains, for example, can be listed below method: after wild type strain is implemented the mutation operation identical with above-mentioned example, select take S-(2-aminoethyl)-Cys (AEC) resistance as index, by becoming the method (referring to Patent Document 1) that has the productive mutant strain of 1B and obtain.Need to prove, whether as described in patent documentation 1, whether having the AEC resistance can by occuring after 24 hours to breed to judge in 30 ℃ of cultivations on the basic nutrient agar that contains 50 μ g/ml AEC.
Perhaps, the more preferably method as give the AEC resistance to wild type strain has the method for having used genetic engineering technique.The method of having used genetic engineering technique is had no particular limits, have (for example) to use the method for the recombinant DNA that in bar-shaped flora, can copy, perhaps make the method for the target gene restructuring in the karyomit(e) by homologous recombination.For example, can obtain S-(2-aminoethyl)-Cys resistance by having mutant aspartokinase gene (sometimes being called as " desensitization type AK gene "), the 311st amino-acid residue in the aminoacid sequence of putting down in writing in this mutant E.C. 2.7.2.4. sequence number 14 is obtained by the amino-acid substitution beyond the Thr.Preferred genetic engineering technique is the method by the AK homologous recombination in the karyomit(e) of bar-shaped flora is recombinated for desensitization type AK gene.In addition, about desensitization type AK gene, can utilize the well-known technological method (for example rite-directed mutagenesis) that imports required sudden change in the AK gene, corresponding test kit is also on sale on market.
In addition, as the mutant strain that the Methionin synthesis capability is improved, except the AEC resistant mutant strain, also can enumerate the homoserine auxotrophic mutant.Wild type strain does not have the homoserine auxotrophy, but as the method that obtains the homoserine auxotrophic strain, can enumerate following methods: identical with above-mentioned example, for example, behind wild type strain enforcement mutation operation, select take the homoserine auxotrophy as index, by becoming the method that has the productive mutant strain of 1B and obtain; Perhaps make the method (patent documentation 1) of the homoserine dehydrogenase activity forfeiture of bar-shaped flora by genetic engineering technique.
In addition, as the more preferably method of giving the homoserine auxotrophy to wild type strain, can enumerate method by inserting other gene in the homoserine dehydrogenase gene (being designated hereinafter simply as HOM) of homologous recombination in the karyomit(e) etc., make thus the method for homoserine dehydrogenase activity forfeiture.Described other gene is had no particular limits.Be preferably the LDC gene, promotor and box gene that more preferably the LDC gene can the constructive expression in wild type strain.
In the present invention, by having cadaverine throughput and the bar-shaped flora that 2,2'-thiobis (ethamine) has a resistance cultivated to make cadaverine described.As cultural method, can use batch culture, feeding culture or cultured continuously.When cultured continuously, preferably carry out cultured continuously by the known method of putting down in writing in (for example) TOHKEMY 2008-104453 communique.
Cultivation substratum as for the manufacture of cadaverine can use the common nutritional medium that contains assimilable Carbon and nitrogen sources and inorganic salts etc.As carbon source, can example such as the carbohydrate such as glucose, fructose, sucrose, maltose, starch hydrolysate, the alcohols such as ethanol, the organic acids such as acetic acid, lactic acid, succsinic acid.As nitrogenous source, can use ammonia, the various inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, volatile salt, ammonium acetate, urea, other nitrogenous compound, and the itrogenous organic substance such as meat extract, yeast extract, corn steep liquor, soya hydrolysate.As inorganic salt, can use potassium phosphate,monobasic, potassium primary phosphate, ammonium sulfate, sodium-chlor, sal epsom, calcium carbonate etc.In addition, as required, can add the micronutrient sources such as vitamin H, VitB1, vitamin B6.These micronutrient sources also can substitute with medium additives such as meat extract, yeast extract, corn steep liquor, casamino acidss.
In addition, also can be to cultivating with the Methionin that adds in advance in the substratum as the cadaverine precursor.If to cultivating with adding in advance Methionin in the substratum, then bar-shaped flora is taken in Methionin in the thalline, lysine decarboxylase changes it into cadaverine take Methionin as matrix, therefore can improve the manufacturing efficient of cadaverine.To cultivating when adding Methionin in advance in the substratum, as cultivating with the lysine concentration in the substratum, not special restriction, but the preferred concentration that can not make a very bad impression to the propagation of bar-shaped flora and can not cause to lysine decarboxylase obstruction particularly is preferably 0.01M to 2M.
The Methionin that adds is preferably 1B.In addition, the Methionin that adds can be episome, also can be lysine salt, but preferred lysine salt, as lysine salt, preferred lysine hydrochloride or derive from the Methionin dicarboxylate of following dicarboxylic acid, as preferred object lesson, can enumerate Methionin adipate, Methionin sebacate, Methionin 1,12-dodecanedicarboxylic acid salt, Methionin succinate, Methionin m-phthalic acid salt, Methionin terephthalate, as preferred object lesson, can enumerate the Methionin adipate.
Culture condition is had no particular limits, under the aerobic conditions such as shaking culture, deep aeration-agitation cultivation, cultivate.Culture temperature is generally 25 ℃ to 42 ℃, is preferably 28 ℃ to 38 ℃.Incubation time is generally 1 day to 6 days.
For the adjusting of cultivating pH, preferably use ammonia when being adjusted to alkalescence, preferably use hydrochloric acid or dicarboxylic acid when being adjusted to acidity, more preferably use dicarboxylic acid.Use these neutralizing agents will cultivate pH regulator to 5 to 8, preferably preferably be controlled at pH6.5 to 7.5.In addition, to the not restriction of state of neutralizing agent, use with gas, liquid, solid or aqueous solution form.The aqueous solution particularly preferably.
The dicarboxylic acid that is preferably used as neutralizing agent is not particularly limited, but preferably, except above-mentioned 2 carboxyls, does not have in fact the dicarboxylic acid of other functional group.Here said functional group be polymerizing polyamide reaction (as reaction conditions, for example, 250 to 270 ℃ of temperature of reaction, pressure 10 is to 20kg/cm
21 to 5 hour reaction times) time, with reactions such as amino or carboxyls, cause the branch of polymkeric substance or make the descend reactive group of (degree of crystallinity is below 80%) of the degree of crystallinity of polymkeric substance, for example, amino or carboxyl just meet this functional group, in addition, acidic-group (sulfonic group, phosphate, phenolic hydroxyl group etc.), basic group (diazanyl etc.), proton polar group (hydroxyl etc.), the group (epoxy group(ing), peroxidation base etc.) with breaking property or other reactive high group (isocyanate base) also meet this functional group.On the other hand, halogenic substituent, aromaticity substituting group, ether, ester group, amide group isoreactivity are low, do not meet said functional group here.
As dicarboxylic acid, more preferably by following general formula (1), (2) or (3) represented dicarboxylic acid.
HOOC-(CH
2)
m-COOH（1）
(in the general formula (1), m=0 to 16).
[Chemical formula 1]
(in the general formula (2), n, o=0 to 16).
[Chemical formula 2]
(in the general formula (3), p, q=0 to 16).
In addition, as dicarboxylic acid, further preferred hexanodioic acid, sebacic acid, 1,12-dodecanedicarboxylic acid, succsinic acid, m-phthalic acid, terephthalic acid.
There is (need to prove, in the present invention they are referred to as " cadaverine ") in cadaverine in the nutrient solution with the episome of cadaverine or the form of cadaverine salt.In order to reclaim the cadaverine in the nutrient solution, at first from nutrient solution, remove bar-shaped flora.At this moment, bar-shaped flora propagation, thereby fermentation fully carries out generating cadaverine, thalline can be separated (as separation method with culture supernatant afterwards, with bacterial sediment remove, centrifugation, membrane filtration separate), perhaps can keep material etc. with thalline separation, maintenance or immobilization from initial just the utilization.The method itself that reclaims cadaverine from the nutrient solution that contains cadaverine of having removed thalline is well-known, for example, and can be as putting down in writing in the TOHKEMY 2009-207495 communique, with form crystallization and the collection of cadaverine dicarboxylate.In addition, also can as putting down in writing in the TOHKEMY 2009-29872 communique, utilize the NF film to make with extra care and collect the episome of cadaverine.In addition, can also be as putting down in writing in the TOHKEMY 2009-28045 communique, by extract with polar organic solvent, and distillation, thereby collect the episome (referring to below with reference to example) of cadaverine.
Embodiment
The below enumerates embodiment and Comparative Examples, and the present invention is described in detail.
The HPLC analytical procedure of reference example 1 cadaverine and lysine concentration
In 25 μ l analytic samples, add 1 of 25 μ l, 4-butanediamine (0.03M) is as internal standard substance, add 150 μ l sodium bicarbonates (0.075M) and 2, the ethanolic soln of 4-dinitrofluorobenzene (0.2M) is mixed, 37 ℃ of lower insulations 1 hour, 50 μ l reaction solns are dissolved in the 1ml acetonitrile, then under the following conditions the supernatant liquor 10 μ l that obtain after centrifugal 5 minutes with the rotating speed of 10,000rpm are carried out HPLC and analyze.
Use pillar: CAPCELL PAK C18(Capital gives birth to hall)
Moving phase: 0.1% (w/w) phosphate aqueous solution: acetonitrile=4.5:5.5
Detect: UV360nm
Reference example 2 has the making (patent documentation 1) of the Corynebacterium glutamicum (TR-CAD1 bacterial strain) of 1B decarboxylase and forfeiture homoserine dehydrogenase activity
(1) HOM gene cloning
In order to make the HOM loss of activity, to cloning corresponding to the gene of 300 amino acid regions that begin from N-terminal.
The base sequence of the HOM gene (registration number BA000036) of registration in the comparable data storehouse (GenBank), synthesized Oligonucleolide primers 5 '-gaagaattctaaacctcagcatctgcccc-3 ' (sequence number 1) and 5 '-gaaggatccaaaggacttgtttaccgacgc-3 ' (sequence number 2).Will be from genomic dna solution Corynebacterium glutamicum ATCC13032, according to conventional methods preparation as amplification template, respectively getting 0.2 μ l packs in the Eppendorf tube of 0.2ml, add all ingredients: the Tris hydrochloride buffer (20mM) of each primer 2 0pmol, pH8.0, Repone K (2.5mM), gelatin (100 μ g/ml), each dNTP(50 μ M), LATaqDNA polysaccharase (2 unit) (precious wine is made system), making total amount is 50 μ l.It is 94 ℃, 30 seconds at the DNA Denaturing, the annealing conditions of primer is 55 ℃, 30 seconds, the lengthening reaction condition of dna primer is under each condition of 72 ℃, 3 minutes, uses the thermal cycler of BioRad company, the polymerase chain reaction (following referred to as the PCR method) of carrying out 30 circulations.In addition, in this reference example, unless specialize, the PCR method is all carried out with this understanding.Carry out electrophoresis by the resulting product of this PCR method with 1% sepharose, from gel, cut out the dna fragmentation of the about 0.9kb that contains the HOM gene, and utilize Gene Clean Kit (BIO101 company system) to make with extra care.This fragment is digested with limiting enzyme EcoRI and BamHI, and the EcoRI-BamHI fragment utilization of resulting 0.9kb connected the precious wine of test kit ver.1(make company's system) be inserted in advance the pHSG298(treasured wine that digest by EcoRI and BamHI and make system) the EcoRI/BamHI gap in, with gained plasmid called after pHOM1.
(2) making of LDC expression cassette
At first, for cloning as the promotor of the kalamycin resistance gene of the promotor of constructive expression LDC in Corynebacterium glutamicum.
The pHSG299(registration number M19415 of registration in the comparable data storehouse (GenBank)) base sequence, synthetic oligonucleotide primer thing 5 '-gaaccgcggcctgaatcgccccatcatcc-3 ' (sequence number 3) and 5 '-gaaccatggccccttgtattactg-3 ' (sequence number 4).Will by take plasmid pHSG299 as amplification template and the resulting product of PCR method take oligonucleotide (sequence number 3), (sequence number 4) as primer sets carry out electrophoresis with 1.0% sepharose, from gel, cut out the dna fragmentation of the 0.3kb of the promoter region that contains kalamycin resistance gene, and utilize Gene Clean Kit to make with extra care.Utilize to connect test kit ver.1 this fragment be inserted into the system in plasmid vector pT7blue(Novagen company) 3 ' end of EcoRV place of incision be added with in the gap of T base, in resulting plasmid, when digesting with restriction enzyme HindIII and SacII, will become the plasmid called after pKMP1 of the single segment of 3.2kb.
Next, carry out the LDC gene cloning.The base sequence of the LDC gene (registration number M76411) of registration in the comparable data storehouse (GenBank), synthetic oligonucleotide primer thing 5 '-gaaccatggacgttattgcaa-3 ' (sequence number 5) and 5 '-gaaccgcggttattttttgctttcttcttt-3 ' (sequence number 6).Will by the PCR method (this PCR method take derive from colon bacillus ATCC10798, with the genomic dna solution of ordinary method preparation as amplification template, take oligonucleotide (sequence number 5), (sequence number 6) as primer sets) resulting product carries out electrophoresis with 1.0% sepharose, from gel, cut out the dna fragmentation of the 2.1kb that contains the LDC gene, and utilize Gene Clean Kit to make with extra care.Utilize connection test kit ver.1 that 3 ' end that this fragment is inserted at the EcoRV of plasmid vector pT7blue place of incision is added with in the gap of T base, in resulting plasmid, when digesting with HindIII and NcoI, will become the plasmid called after pCADA of the single segment of 4.0kb.
At last, with HindIII and NcoI digestion pKMP1, its product is carried out electrophoresis with 1.2% sepharose, from gel, cut out the dna fragmentation of the 0.3kb of the promoter region that contains kalamycin resistance gene, and utilize Gene Clean Kit to make with extra care.Utilize to connect in the HindIII/NcoI gap that HindIII-NcoI fragment that test kit ver.1 will so obtain is inserted in advance the pCADA that was digested by HindIII and NcoI, with gained plasmid called after pTM100.
(3) making of HOM gene disruption and LDC expression vector
Digest pTM100 with SacII, its product is carried out electrophoresis with 1.0% sepharose, from gel, cut out the dna fragmentation of the 2.4kb that contains the LDC expression cassette, and utilize Gene Clean Kit to make with extra care.Utilize to connect in the SacII gap that SacII fragment that test kit ver.1 will so obtain is inserted in advance the pHOM1 that was digested by SacII, with gained plasmid called after pTM101.
(4) pTM101 is to chromosomal integration
By electroporation ［ FEMS Microbiology Letters, 65, p.299 (1989) ］ to Corynebacterium glutamicum ATCC13032(hereinafter to be referred as the ATCC13032 bacterial strain) in import plasmid pTM101, at the LB(Tryptones (10g/l) that is added with kantlex (25 μ g/ml) (Bacto company system), yeast extract (5g/l) (Bacto company system), sodium-chlor (10g/l)) nutrient agar chooses.
From the transformant of choosing like this, prepare genomic dna solution with ordinary method.Take this genomic dna as template, carry out PCR take oligonucleotide (sequence number 5) (sequence number 6) as primer sets, products therefrom is carried out electrophoresis with 1.0% sepharose, observe the single band of 2.1kb this moment.Can confirm thus, for selected transformant, the LDC gene is inserted in the HOM locus.This transformant called after Corynebacterium glutamicum TR-CAD1(is designated hereinafter simply as the TR-CAD1 bacterial strain).
Confirmed that 13032 bacterial strains do not have the LDC activity, and the TR-CAD1 bacterial strain has the LDC activity.In addition, 13032 bacterial strains have the HOM activity, and the TR-CAD1 bacterial strain has been lost the HOM activity.13032 bacterial strains have no lack of homoserine, and the TR-CAD1 bacterial strain lacks homoserine.Can make thus the Corynebacterium glutamicum TR-CAD1 bacterial strain with LDC activity and forfeiture HOM active (homoserine auxotrophy).
Reference example 3 has the 1B decarboxylase, have the homoserine defective and have the making (patent documentation 1) of Corynebacterium glutamicum (TR-CAD2) bacterial strain of S-(2-aminoethyl)-Cys resistance
The making of (1) desensitization type AK gene
Make desensitization type AK gene in order in the AK gene, to import sudden change, the AK gene is cloned.
The AK gene of registration in the reference database (GenBank) (registration number: base sequence BA000036), synthetic oligonucleotide primer thing 5 '-acagaattcgtggccctggtcgtacagaa-3 ' (sequence number 7) and 5 '-catctcgagttagcgtccggtgcctgcat-3 ' (sequence number 8).To carry out electrophoresis with 1.0% sepharose by the resulting product of PCR method (this PCR method is to derive from genomic dna solution Corynebacterium glutamicum ATCC13032, according to conventional methods preparation as amplification template, take oligonucleotide (sequence number 7), (sequence number 8) as primer sets), from gel, cut out the dna fragmentation of the about 1.3kb that contains the AK gene, and utilize Gene Clean Kit to make with extra care.This fragment is digested with EcoRI and XhoI, utilize connecting test kit ver.1 is inserted in advance the pHSG396(treasured wine that digest by EcoRI and XhoI with the EcoRI-XhoI fragment of resulting 1.3kb and makes system) the EcoRI/XhoI gap in, with gained plasmid called after pAK1.
Next, the 931st to the 933rd acc(Thr for the AK gene that makes the clone) sport atg(Ile), synthesized Oligonucleolide primers 5 '-cgacatcatcttcacctgcc-3 ' (sequence number 9) and 5 '-ggcaggtgaagatgatgtcg-3 ' (sequence number 10).Will be take pAK1 as amplification template, take oligonucleotide (sequence number 9), (sequence number 10) is as primer sets and use QuickChange Site-Directed Mutagenesis Kit(ス ト ラ タ ジ ー Application Co., Ltd. system) the plasmid called after pTM102 that obtains.By ordinary method the AK gene among this pTM102 is screened, can confirm this moment: according to purpose, from the 931st to the 933rd acc(Thr) sport atg(Ile), thereby made desensitization type AK gene.
(2) pTM102 is to chromosomal integration
The pFK398(registration number D29826 of registration in the reference database (GenBank)) base sequence, the Oligonucleolide primers 5 of synthesizing chloramphenicol resistant gene '-acggtcgactcgcagaataaataaatcctggtg-3 ' (sequence number 11) and 5 '-atgaggcctgagaggcggtttgcgtattgga-3 ' (sequence number 12).
In the TR-CAD1 bacterial strain, import plasmid pTM102 by electroporation, and choose at the LB nutrient agar that is added with kantlex (25 μ g/ml) and paraxin (10 μ g/ml).
From the transformant of choosing like this, prepare genomic dna solution with ordinary method.Take this genomic dna as template, carry out PCR take oligonucleotide (sequence number 11) (sequence number 12) as primer sets, products therefrom is carried out electrophoresis with 1.0% sepharose, observe the single band of 1.0kb this moment.Can confirm thus, for selected transformant, chloramphenicol resistance gene is inserted in the AK locus.This transformant called after Corynebacterium glutamicum TR-CAD2(is designated hereinafter simply as the TR-CAD2 bacterial strain).
(3) import the affirmation of desensitization type AK gene on the karyomit(e) of TR-CAD2 bacterial strain
The base sequence of the AK gene (registration number BA000036) of registration in the reference database (GenBank), synthetic than the N-terminal of AK be in the Oligonucleolide primers 5 of the 0.1kb of upstream '-ttggaacgcgtcccagtggc-3 ' (sequence number 13).
From the TR-CAD2 bacterial strain, prepare genomic dna solution with ordinary method.Take this genomic dna as template, carry out the PCR method take oligonucleotide (sequence number 12) (sequence number 13) as primer sets, products therefrom is carried out electrophoresis with 1.0% sepharose, from gel, cut out the dna fragmentation of the 3.1kb that contains AK gene and chloramphenicol resistance gene, and utilize Gene Clean Kit to make with extra care.Utilize connection test kit ver.1 that 3 ' end that this fragment is inserted at the EcoRV of plasmid vector pT7blue place of incision is added with in the gap of T base, with gained plasmid called after pAK2.By ordinary method the AK gene among this pAK2 is screened, can confirm to contain desired sudden change this moment, thereby can confirm and can import the desensitization type AK gene that can express on the karyomit(e) of TR-CAD2 bacterial strain.Can make thus and have the LDC activity, lose HOM active (homoserine auxotrophy) and be the Corynebacterium glutamicum of AEC resistance (TR-CAD2 bacterial strain).
Embodiment 1 has the ability of producing cadaverine and 2,2'-thiobis (ethamine) is had the obtaining of bar-shaped flora of resistance
With the Corynebacterium glutamicum (TR-CAD1 bacterial strain) that obtains in the above described manner and Corynebacterium glutamicum (TR-CAD2 bacterial strain) respectively in the BHI(of 5ml brain heart infusion, Brain Heart Infusion) carries out the front cultivation of a whole night in the liquid nutrient medium, 2.5ml in the nutrient solution before the gained is inoculated in the new BHI liquid nutrient medium, be cultured to turbidity (A600) and reach 1～2, wherein said Corynebacterium glutamicum (TR-CAD1 bacterial strain) is owing to having the 1B decarboxylase, and lose homoserine dehydrogenase activity and become the homoserine auxotrophic mutant; Described Corynebacterium glutamicum (TR-CAD2 bacterial strain) becomes the homoserine auxotrophic mutant owing to having the 1B decarboxylase and losing homoserine dehydrogenase activity, and it has S-(2-aminoethyl)-Cys resistance (AEC resistance).After the cultivation, with Tris-toxilic acid buffer solution for cleaning thalline 2 times, the thalline that cleaned is hanged turbid in the Tris-of 12ml toxilic acid damping fluid, with the mode packing of suspension liquid with every test tube 900 μ l, and add respectively the NTG solution of 640 μ g/ml.Mixing solutions is 30 ℃ of lower thermal agitations 40 minutes, then outstanding turbid in the BHI of 2ml substratum, and with ice-cooled.After suspension liquid usefulness Tris-toxilic acid buffer solution for cleaning 2 times, add the BHI substratum of 50ml, 28 ℃ of lower cultivations a whole night.With nutrient solution Tris-toxilic acid buffer solution for cleaning 2 times, with having added 2 of 180mM, 220mM, 250mM, 300mM or 400mM, culture medium culturing is after 2～3 days shown in the table 1 of 2'-thiobis (ethamine), acquisition has formed the thalline of bacterium colony as 2,2'-thiobis (ethamine) resistant strain is respectively TR-CADA1-C, TR-CADA1-0～6 bacterial strains, TR-CADA2-C, TR-CADA2-0～6 bacterial strains (table 2).Further with the resistant strain that obtains with the culture medium culturing of obtaining this resistant strain 3 days, confirm to have formed flora, that is, confirm to have given 2,2'-thiobis (ethamine) resistance.
Need to prove, TR-CADA1-6 bacterial strain, TR-CADA2-5 bacterial strain are deposited in the Japanese national technological assessment institute (independent independent administrative institution System product Evaluation value skill Intraoperative base Disk Machine Agencies, NITE) by the world to accept numbering NITE BP-1002, NITE BP-1003 respectively.
[table 2]
Embodiment 2,3 and Comparative Examples 1,2
Utilize TR-CADA1-1～6 bacterial strains (embodiment 2), TR-CADA2-1～6 bacterial strains (embodiment 3), TR-CADA1 bacterial strain (Comparative Examples 1), TR-CADA1-0 bacterial strain (Comparative Examples 2), TR-CADA2(Comparative Examples 3), TR-CADA2-0(Comparative Examples 4) carry out cadaverine fermentation.Each bacterial strain of 5mL BHI inoculation of medium 1 platinum loop after the sterilization 30 ℃ of lower vibrations 24 hours, carries out preliminary preculture.This preliminary preculture liquid all is inoculated in the substratum identical with preliminary preculture of 50ml, under 30 ℃, the condition of amplitude 30cm, 120rpm, cultivates 24 hours to carry out preculture.Next, preculture liquid all is inoculated into shown in the table 3 of 950ml in the MMP substratum, air after the sterilization is ventilated with the flow velocity of 0.07vvm, simultaneously under 30 ℃, rotating speed of agitator 800rpm, pH are adjusted to 6.7 condition, carry out 50 hours cultivation.Neutralizing agent uses aqueous sulfuric acid (3M) and ammoniacal liquor (3M).
[table 3]
After cultivating end, at 4 ℃, 8, centrifugation is 10 minutes under the 000rpm, removes thus thalline, and reclaims culture supernatant.Utilize HPLC that the cadaverine in this culture supernatant and Methionin are analyzed.In addition, glucose concentration determination has used " Glucose Test Wako C " (registered trademark) (with light Pure medicine society system).Calculate cadaverine to sugared yield (the glucose weight of the cadaverine weight of generation/consumption) * 100 (%)), the result is as shown in table 4.
[table 4]
The result shows, when embodiment 2 being compared respectively with Comparative Examples 1, Comparative Examples 2 and with embodiment 3 and Comparative Examples 3, Comparative Examples 4 respectively relatively the time, by maternal plant being given 2,2'-thiobis (ethamine) resistance, improved cadaverine accumulate concentration and to sugared yield.
Reclaiming cadaverine by the gained culture supernatant can be undertaken by various known methods.The below enumerates preferred known recovery method.
Reference example 4 reclaims cadaverine from culture supernatant
Adding the sodium hydroxide of 5N in the culture supernatant of the cadaverine fermented liquid that obtains by aforesaid method, is 13 with pH regulator.Then, make its by nanofiltration membrane to remove the remaining thalline of inorganic salt composition and trace, reclaim the nanofiltration permeate.Then, make the nanofiltration permeate of recovery by reverse osmosis membrane, being concentrated into cadaverine concentration becomes about 18 % by weight, and reclaims.Then, the concentrated solution that reclaims is further carried out concentrating under reduced pressure by rotatory evaporator etc., thus except anhydrating, thereby obtain the cadaverine aqueous solution about 50 % by weight.Next, add the chloroform of equivalent, make cadaverine be extracted into chloroform mutually in.At last, this chloroform is carried out underpressure distillation (30mmHg, 80 ℃) mutually, isolate thus free cadaverine.
Industrial applicibility
By the present invention, can be at industrial manufacturing cadaverine.
[preserving number]
NITE?BP-1002
NITE?BP-1003
Filled in by the office of accepting
Filled in by international office
Claims (5)
1. the manufacture method of a cadaverine comprises cultivating to have the ability of producing cadaverine and the bar-shaped flora that 2,2'-thiobis (ethamine) is had resistance.
2. the manufacture method of cadaverine according to claim 1, described bar-shaped flora is to 2 more than the 250mM, and 2'-thiobis (ethamine) has resistance.
3. the manufacture method of cadaverine according to claim 1 and 2, described bar-shaped flora has the lysine decarboxylase activity.
4. the manufacture method of each described cadaverine in 3 according to claim 1, the group that the free corynebacterium of described corynebacterium mass selection and brevibacterium sp consist of.
5. the manufacture method of each described cadaverine in 4 according to claim 1, described bar-shaped flora has homoserine auxotrophy and/or S-(2-aminoethyl)-Cys resistance.
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| US9919996B2 (en) | 2013-01-28 | 2018-03-20 | Cathay R&D Center Co., Ltd. | Preparation of cadaverine |
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| CN101240258A (en) * | 2007-02-01 | 2008-08-13 | 赢创德固赛有限责任公司 | Process for production of cadaverine by fermentation |
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| BRPI0707027B1 (en) * | 2006-02-24 | 2022-08-16 | Toray Industries, Inc | METHOD OF MANUFACTURING A CHEMICAL PRODUCT AND CONTINUOUS FERMENTATION APPLIANCE |
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| US20120288901A1 (en) | 2007-02-19 | 2012-11-15 | Oskar Zelder | Method of Producing Methionine in Corynebacteria by Over-Expressing Enzymes of the Pentose Phosphate Pathway |
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| JP4983861B2 (en) | 2009-06-15 | 2012-07-25 | 東レ株式会社 | Polyamide made from cadaverine dicarboxylate |
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Also Published As
| Publication number | Publication date |
|---|---|
| BR112013014197A2 (en) | 2017-06-06 |
| EP2650374A1 (en) | 2013-10-16 |
| EP2650374B1 (en) | 2018-05-30 |
| BR112013014197B1 (en) | 2020-11-17 |
| JPWO2012077741A1 (en) | 2014-05-22 |
| US9080190B2 (en) | 2015-07-14 |
| WO2012077741A1 (en) | 2012-06-14 |
| EP2650374A4 (en) | 2015-07-08 |
| CN103328643B (en) | 2015-08-19 |
| KR20130135859A (en) | 2013-12-11 |
| US20130323800A1 (en) | 2013-12-05 |
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